Extracellular vesicles released from microglia after palmitate exposure impact brain function

Dietary patterns that include an excess of foods rich in saturated fat are associated with brain dysfunction. Although microgliosis has been proposed to play a key role in the development of brain dysfunction in diet-induced obesity (DIO), neuroinflammation with cytokine over-expression is not always observed. Thus, mechanisms by which microglia contribute to brain impairment in DIO are uncertain. Using the BV2 cell model, we investigated the gliosis profile of microglia exposed to palmitate (200 µmol/L), a saturated fatty acid abundant in high-fat diet and in the brain of obese individuals. We observed that microglia respond to a 24-hour palmitate exposure with increased proliferation, and with a metabolic network rearrangement that favors energy production from glycolysis rather than oxidative metabolism, despite stimulated mitochondria biogenesis. In addition, while palmitate did not induce increased cytokine expression, it modified the protein cargo of released extracellular vesicles (EVs). When administered intra-cerebroventricularly to mice, EVs secreted from palmitate-exposed microglia in vitro led to memory impairment, depression-like behavior, and glucose intolerance, when compared to mice receiving EVs from vehicle-treated microglia. We conclude that microglia exposed to palmitate can mediate brain dysfunction through the cargo of shed EVs. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-024-03168-7.


Palmitate preparation
A stock solution of sodium palmitate was prepared by conjugation with bovine serum albumin (BSA).Briefly, 4.54 g of fatty acid-free BSA (#A7030, Sigma-Aldrich, St. Louis, MO-USA) was dissolved at 36ºC in 16 mL of 150 mmol/L NaCl, and 61.2 mg of sodium palmitate (Sigma-Aldrich #P9767) was dissolved at 71ºC in 4 mL of 150 mmol/L NaCl.The palmitate solution was then slowly added to the BSA solution while stirring, filtered with a sterile 22-µm polyvinylidene fluoride filter (Sigma-Aldrich #LGVV255F), and frozen at -20 ºC in 11 mmol/L palmitate aliquots.

ECAR assay
Cells were incubated in assay medium without glucose or pyruvate (sodium bicarbonate and FBS were absent) in atmospheric air at 37 °C.Baseline ECAR was measured after addition of 10 mmol/L glucose.The conversion of glucose to pyruvate, and production of lactate that is released with protons leads to medium acidification that can be used as surrogate of glycolysis.
NMR spectra were acquired on a Avance III HD 600 MHz spectrometer with a standard TCI cryoprobe (Bruker Nordic, Solna, Sweden).Solvent-suppressed 1 H-NMR spectra were acquired with the ZGPR pre-saturation pulse sequence with spectral width of 9 kHz, 3 s acquisition time, a relaxation delay of 22 s, and 24 scans per cell extract. 1 H-decoupled 13 C-NMR spectra were acquired using the ZGPG30 sequence with 30 kHz spectral width, 2 s acquisition time, and a relaxation delay of 2 s.To achieve adequate signal-to-noise ratio, 13 C spectra were recorded with at least 30,000 scans.
Samples were analyzed in an Orbitrap Eclipse Tribrid mass spectrometer coupled with an Ultimate 3000 RSLCnano system (ThermoFischer).The HPLC used a two-column setup: peptides were loaded into an Acclaim PepMap 100 C18 pre-column (75 μm x 2 cm; ThermoFischer) and then separated with the flow rate 300 nL/min in an EASYspray column (75 μm x 25 cm, nanoViper, C18, 2 μm, 100 Å; ThermoFischer).The column temperature was set 45 °C.Peptides were eluted with a nonlinear gradient using 0.1%(v/v) formic acid in water as solvent A, and 0.1%(v/v) formic acid in 80%(v/v) acetonitrile as solvent B. Solvent B was maintained at 2% during 4 minutes, increased to 25% during 100 minutes, to 40% during 20 minutes, to 95% during 1 minute, and finally kept at 95% for 5 min to wash the column.
Samples were analyzed with the positive data-dependent acquisition (DDA) mode.The full MS resolution was set to 120,000 at normal mass range, and the automatic gain control target (AGC) was set to standard with the maximum injection time to auto.The full mass range was set 350-1400 m/z.Precursors were isolated with the isolation window of 1.6 m/z and fragmented by HCD with the normalized collision energy of 30.MS 2 was detected in the Orbitrap with the resolution of 15,000, and AGC and maximum injection time were set to standard and 50 ms, respectively.
The raw DDA data were analyzed with Proteome Discoverer 2.5 Software (ThermoScientific), and the peptides were identified using SEQUEST HT against the UniProtKB Mouse database (UP000000589) with the following parameters applied: cysteine carbamidomethylation as static modification, and N-terminal acetylation and methionine oxidation as dynamic modification.Precursor tolerance was set to 10 ppm, and fragment tolerance was set to 0.05 ppm.Up to 2 missed cleavages were allowed.Percolator false discovery rate (FDR) was used for peptide validation at a q-value below 0.01.The extracted chromatographic intensities were used to compare peptide abundance across samples.

Injection of EVs in the lateral ventricle
Mice were anesthetized with isoflurane (induction with 5%; maintenance with 2-3% in air), and their heads were fixed at a 45° angle in a stereotactic frame (Kopf Instruments, Tujunga, CA-USA) and body placed on a heating pad.After craniotomy under magnification, a glass micropipette (diameter 20-40 μm) was used to deliver 500 ng protein from fresh BV2-derived EVs to the lateral ventricle (anterior/posterior, 0.34 mm from bregma; medial/lateral, 1.0; dorsal/ventral, -2.2 mm from the skull; injection site confirmed in pilot experiments injecting trypan blue).EVs were delivered in multiple microinjections over 3 minutes using air pressure from a PLI-100A Pico-Injector (Harvard Apparatus, Cambridge, UK), and were allowed to diffuse during 4 minutes before the needle was withdrawn.After, animals received subcutaneous saline for hydration (1 mL), and 5 mg/kg Bupivacaine (Marcain, Aspen Nordic, Ballerup, Danmark) for pain relief upon recovery.

Table S1 .
Results from ANOVA statistics

Table S2 .
Differential expression testing results following comparison of proteomes of EVs released from microglia exposed to vehicle, palmitate and LPS.

Table S3 .
Proteomic data from 3 samples of EVs released from microglia exposed to vehicle, palmitate and LPS.